Standard hemodialysis readily removes molecules (e.g., urea, raffinose, and potassium) in the body fluids which are free and not bound to other molecules like proteins. However, molecules such as jaundice-causing bilirubin and tranquilizer diazepam can not be removed from body fluids by standard hemodialysis because such molecules, though small, are bound to larger molecules like albumin (MW 69,000) and other proteins present in the body fluids: standard hemodialysis membranes are not permeable to albumin or large proteins because their pores are too tiny to admit them (FIG. 1); consequently molecules bound to large proteins remain stuck to the proteins and do not cross the membrane.
Some years ago, I invented a hybrid bio-artificial liver (U.S. patent Ser. No. 3,734,851) which works on the principle of flowing body fluid across one side of a semi-permeable membrane while keeping living liver cells (hepatocytes) proximate opposite side of said membrane. The membrane was physiologically semi-permeable in that the porosity allowed cross-over of molecules such as nutrients, toxins, proteins, and hepatocyte-secretory products, but prevented the cross-over of cells such as lymphocytes in the body fluid which can immunologically reject and kill the hepatocytes. Protein-bound toxins that are normally removed by the liver are also removed by my bio-artificial liver.
However, it became advantageous to use animal hepatocytes to treat human beings because animal hepatocytes are more readily available in quantity and are generally cheaper than human hepatocytes. Because it can be disadvantageous to allow contamination of human body fluid with animal proteins, it was felt best to use a semi-permeable membrane that would not allow the cross-over of proteins like albumin. It was discovered, however, that just any randomly selected membrane impervious to proteins would not work well in my bio-artificial liver because protein-bound toxins and substances in the body fluid remained unremoved. Work was done to figure out how to remove protein-bound substances while still using semi-permeable membrane that would not allow the cross-over of proteins.
While conducting extensive studies over many years on the removal of protein-bound substances by hepatocytes, some other interesting observations were made which became the bases of the invention described herein.
If one flows body fluid containing bilirubin molecules that are bound to albumin simply across the surface of hepatocytes extracted from liver tissue, one is surprised to find that the hepatocytes do not remove the bilirubin molecules as well as would be commonly expected. Those skilled in the art had always believed that one can create a bio-hybrid artificial liver by simply passing body fluid across the surfaces of hepatocytes which are isolated from liver and which are attached to a solid surface such as a membrane or beads. As the inventor of the first practical bio-artificial liver (cited above), I also believed the same, that the only ingredients needed for a fully functional bio-artificial liver were 1) live hepatocytes and 2) flowing body fluid that can feed nutrients and oxygen to the liver cells. While my bio-artificial liver of the aforementioned patent employed a semi-permeable membrane interposed between the flowing body fluid and the hepatocytes proximate the membrane, I had intended that the purpose of the membrane was purely to prevent the flowing body fluid from washing away the hepatocytes and to prevent immunologic cells in the body fluid from attacking and destroying the hepatocytes which may be invariably immunologically incompatible with the patient from which the body fluid flowed. For this reason, this semi-permeable membrane was exactly that: semi-permeable. I specified that the membrane be permeable to molecules but that it allows no immunologic cells to cross the pores of the membrane even over time slowly.
As other technologies developed so that 1) hepatocytes can be attached so securely to a solid surface so as to make their washing away no longer a problem, and 2) immunologic rejection of hepatocytes can be prevented without a semi-permeable membrane, I began to envision a bio-artificial liver using no semi-permeable membrane where hepatocytes would be bathed directly in the flowing body fluid. As we began to have success in growing human hepatocytes in culture, it became possible to consider a bio-artificial liver using no semi-permeable membrane which would be no longer necessary to block contamination of the treated body fluid with animal proteins from animal hepatocytes. It was expected that such a bio-artificial liver would be more efficient since molecules to be treated in the body fluid did not have the obstacle of having to cross the membrane to be taken up by the hepatocytes. In fact, others (B. Eiseman, L. Norton, N. C. Kralios, Surg. Gyn. Obst. 142:21-28, Jan. 1976; J. Uchino, et al. Proc. Am. Soc. Artif. Intern. Organs 34:972-977, 1988; K. Yanagi, et al. Proc. Am. Soc. Artif. Intern. Organs 36:M727-M729, 1990; T. Soyer et al. Annals of Surg. 177:393-401, 1973) proposed bio-artificial livers in which hepatocytes were directly bathed by flowing body fluids such as plasma from a continuous plasmapheresis device. Although it has not been publicly reported, nothing has yet come of bio-artificial livers not using an interposing semi-permeable membrane between hepatocytes and body fluid.
In light of the the discoveries I have been making over the past twenty years pertaining to the removal of protein-bound substances from plasma by hepatocytes, it is now not surprising that a bio-artificial liver does not efficiently remove protein-bound bilirubin if one simply flows body fluid across the surfaces of hepatocytes.
What I have learned mostly through experimentation is that it is necessary to interpose a layer of substantially increased albumin concentration between the flowing body fluid and the hepatocytes.